| It has been well established that articular cartilage has a limited capacity for self-repair. Cartilage lesions, whenever resulting from trauma or due to the tumor, often fail to heal spontaneously and may lead to progressive destruction of the joint and the onset of osteoarthritis. Various methods have been developed to augment its healing response, for example, microfracture, subchondral drilling, perichondrial/periosteal grafts. But these efforts generally seem to be quite limited. Recently, the rapid development of tissue engineering has brought great chance for the cartilage repair. Based on the discipline of cell biology, biomaterial science and engineering, the aim of tissue engineering is to restore the structure and function of tissues lost in the injury or disease. Till now, the development of novel scaffold is the main task in cartilage tissue eingineering. In this study, a novel nano-amorphous calcium phosphate/Poly L-lactic acid (ACP/PLLA) scaffold was developed by a thermally induced phase separation technique. And this tissue engineered scaffold incorporated with basic fibroblast growth factor (bFGF) was introduced to repair articular cartilage defect.Part I The synthesis and characterisation of ACP/PLLA hybrid materialObjective: To characterize the novel ACP/PLLA composite and evaluate the feasibility of this material as a scaffold for cartilage tissue engineering. Methods: The novel ACP/PLLA scaffold was developed by the thermally induced phase separation technique. The morphology, porous size, porosity and the mechanical properties of the scaffold was evaluated. The morphology changes were also observed by scanning electron microscope (SEM) when ACP/PLLA was soaked in PBS solution. Furthermore, when the growth factor bFGF was incorporated into ACP/PLLA, the controlled release of bFGF was examined. Results: The novel ACP/PLLA material was a porous three-dimensional scaffold with the pore size of around 100-200μm, porosity of 91% and elastic modulus of 3.46Mpa. When the scaffold was soaked in PBS solution, ACP particles coated on the PLLA pore walls could experience a fast phase transformation and morphological variation to flake-like crystallites. When the growth factor bFGF was incoporated into the scaffold, bFGF could release in a substained manner. Conclusion: The novel ACP/PLLA hybrid material can act as an ideal scaffold for cartilage tissue engineering and vehicle for growth factor.Part II The isolation, culture of bMSCs and the biocompatibility of ACP/PLLA material with bMSCsObjective: To investigate the method of isolation, culture of bone marrow-derived mesenchymal stem cells (bMSCs) from rabbits and evaluate their biocompatibility with ACP/PLLA scaffold. Methods: bMSCs were extracted from the bone marrow in rabbits by density gradient centrifugation method and cultured in vitro regularly. When bMSCs reached confluence, they were subcultured. The cells of the 3nd passage were seeded into ACP/PLLA and PLLA scaffolds to investigate the cell attachment and proliferation by MTT assay. The cell morphology was observed by SEM and Confocal Laser Scanning Microscope (CLSM). Results: The obtained primary bMSCs began to adhere at 3 days. Generally, it would take 14 days for the primary cells to reach confluence, and only 7 days for the passaged cells. When bMSCs were seeded into scaffolds, the cell attachment rate in ACP/PLLA was 77.8%, which was statistically higher than that in PLLA scaffold. The cell proliferation assay showed that the cell number in ACP/PLLA group and monolayer group was much higher than that in PLLA group (P<0.05) at 3 and 6 days. Thereafter, the cell proliferation rate in monolayer group slowed down, while the cells in scaffolds still increased rapidly. At 10 days, the cell number in ACP/PLLA group was higher than that in PLLA group (P<0.05). The SEM and CLSM observation further confirmed that the cell density in ACP/PLLA group was obviously higher than in PLLA group. Additionally, the SEM observation revealed that bMSCs adhered tightly onto the pore surface and began to secret the matrix. Conclusion: bMSCs can be obtained from bone marrow by the density gradient centrifugation method. Compared with PLLA scaffold, ACP/PLLA scaffold can promote the cell attachment and proliferation, thus be a more superior scaffold for cartilage tissue engineering.Part III The experimental study of osteochondral repair by the combination of ACP/PLLA scaffold and bFGFObjective: To investigate the efficacy of ACP/PLLA scaffold combined with bFGF to repair osteochondral defects in a rabbit model. Methods: Osteochondral defects (4mm in diameter and 5mm in depth) were created in the medial femoral condyles in 21 skeletally mature rabbits. The defects were either implanted with ACP/PLLA loaded with bFGF, PLLA and bFGF composite or left untreated. The rabbits were sacrificed at 4 and 12 weeks after implantation. Samples were evaluated by macroscopic and histological examination. The gene expression of the newly formed tissue was also detected by RT-PCR analysis. Results: 4 weeks after surgery, the defects were filled with fibrous or immature repair tissue in ACP/PLLA/bFGF and PLLA/bFGF groups. No obvious cartilage tissue was observed in both groups. At 12 weeks, a typical layer of hyaline cartilage was formed in the repair tissue in ACP/PLLA/bFGF group. A continuous layer of subcondral bone was also detected below the cartilage layer. However, the newly formed tissue in PLLA/bFGF group was mainly fibrocartilage with no obvious subcondral bone formed. In control group, the defect was still remained and covered by a thin layer of fibrous tissue whenever in 4 and 12 weeks, indicating the poor self-repair ability of cartilage defects. RT-PCR analysis showed that high levels of type II collagen (Col II) and aggrecan message were detected in repair tissue in ACP/PLLA/bFGF group. In contrast, only a small amount of Col II appeared in PLLA/bFGF group with no aggrecan gene expression detected. Conclusion: The osteochondral defects can be successfully repaired by the combination of ACP/PLLA and bFGF.
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